The generalized loss, as well as the local destruction of bone that attends such common skeletal diseases as osteoporosis and rheumatoid arthritis is largely the result of increased osteoclastic activity. The mechanism whereby osteoclasts resorb bone, however, are unknown. Preliminary studies, using microsampling techniques, indicate that, in the microenviroment of the bone resorbing cell-bone matrix interface, the pH is acidic, (pH about 5.21) compared to the extracellular fluid bathing bone, not in contact with cells (pH about 7.33). These preliminary data suggest that the quality as well as the quantity of the fluid secreted from resorbing cells may play a role in the bone resorptive process. I now plan to expand this study and propse to use an in vitro bone resorbing system consisting of isolated osteoclasts and multinucleated giant cells to measure: the pH changes; activity of degradation enzymes; and levels of electrolytes in the extracellular fluid at the resorbing cell-bone matrix interface. This will be possible to do using micropuncture techniquqee and ultramicroanalytical methods. Specific areas to be covered are: (1) further development of an in vitro bone resorbing sysytem that utilizes isolated chick osteoclasts and rat microphage polykaryons (multinucleated giant cells); (2) modify existing micropuncture methodology for mocrosdampling the fluid at the resorbing cell-bone matrix interface; (3) determine in this resorbing fluid the: pH by micro pH electrode; activity of degradative enzymes (acid phosphatase, cathepsin B, and collagenase) by substrate hydrolysis; and calcium, phosphorus , and other electrolytes by electron probe microanalysis; (4) characterize the changes with time, in the microenvironment of these cells induced by known modulators of bone resorption (PTH, calcitonin, cortisol, prostaglandins, and vitamin D metabolites); and (5) correlate the quantitative morphologic changes if these cells to changes in resorptive activity using scanning and transmission electron microscopy.